In order to understand the development and functioning of the thymus, both in terms of T cell differentiation and stromal cell environmental support, we have undertaken a molecular approach to identify genes that are uniquely expressed in this organ. We have created an anchored RT-PCR based cDNA library from day 14 fetal thymuses after culturing them in deoxyguanosine and treating them with an anti-CD45 antibody to deplete the cell population of hematopoietic cells. The cDNA library was subtracted with poly A+ RNA prepared first from a fibroblast cell line and then from whole spleen. Three prime sequencing of 250 random cDNAs revealed 137 which were not in the databases of known sequences. The novel cDNAs were then screened by Northern blotting for expression in various tissues and in a set of SV40 transformed thymic epithelial cell lines. Four genes were selected for further analysis because they were limited in their expression to the thymus or to one of the stromal cell lines. All four were completely sequenced by obtaining full length cDNA clones from a SCID thymus library.During the past year the lab has made progress in understanding the structure and function of two of these genes. 1)The LamC2 gene, encoding the gamma 2 chain of laminin 5, an epithelial cell-specific extracellular matrix protein, was highly expressed in SCID thymus and in a nurse cell line, but not in other thymic epithelial cell lines. In situ hybridization and immunohistochemical staining revealed laminin 5 expression mostly in the subcapsular region of the adult thymus where nurse cells are located. Addition to fetal thymic organ cultures of a cell adhesion-blocking monoclonal antibody directed against the alpha 3 chain of laminin 5 interrupted T cell development. There was a 40% reduction in the total yield of thymocytes, and the most profound decrease (75-90%) was seen in the CD25+CD44+ and CD25+CD44- subsets of the CD4-CD8- double negative(DN) fraction. Most of the surviving DN thymocytes expressed Sca-1, an early developmental antigen, and there were significant increases in both the number of cells with CD69 expression and in the fraction of annexin V stained cells, markers associated with the induction of apoptosis. None of these changes were observed with a nonblocking anti-laminin alpha 3 chain monoclonal antibody. These results suggest that the interaction between DN thymoctyes and laminin 5 made by subcapsular epithelial cells is required for the survival and differentiation of early mouse thymocytes. 2) Spatial, a gene with weak homology to transcription factors containing a POU domain and localized to the nucleus using a green fluorescent protein tag, was deleted by insertional mutagenesis. Young homozygous gene-targeted mice showed generally normal thymic development. With age, however, the DN thymocyte subpopulation appeared to increase in size and all subsets within it were increased 2 to 4 fold. The biological impact of this change in T cell development with age is currently being investigated.